Cultured HT-29 Cells Research Articles

Studies on the Probiotic, Adhesion, and Induction Properties of Artisanal Lactic Acid Bacteria: to Customize a Gastrointestinal Niche to Trigger Anti-obesity Functions.

The primary goals of this work are to explore the potential of probiotic lactic acid bacteria's (LAB) mucin/mucus layer thickening properties and to identify anti-obesity candidate strains that improve appropriate habitat for use with the Akkermansia group population in the future. The HT-29 cell binding, antimicrobial properties, adhesion to the mucin/mucus layer, growth in the presence of mucin, stability during in vitro gastrointestinal (GI) conditions, biofilm formation, and mucin/mucus thickness increment abilities were all assessed for artisanal LAB strains. Sixteen LAB strains out of 40 were chosen for further analysis based on their ability to withstand GI conditions. Thirteen strains remained viable in simulated intestinal fluid, while most showed high viability in gastric juice simulation. Furthermore, 35.9-65.4% of those 16 bacteria adhered to the mucin layer. Besides, different lactate levels were produced, and Streptococcus thermophilus UIN9 exhibited the highest biofilm development. In the HT-29 cell culture, the highest mucin levels were 333.87µg/mL with O. AK8 at 50mM lactate, 313.38µg/mL with Lactobacillus acidophilus NRRL-B 1910 with initial mucin, and 311.41µg/mL with Lacticaseibacillus casei NRRL-B 441 with initial mucin and 50mM lactate. Nine LAB strains have been proposed as anti-obesity candidates, with olive isolates of Lactiplantibacillus plantarum being particularly important due to their ability to avoid mucin sugar consumption. Probiotic LAB's attachment to the colonic mucosa and its ability to stimulate HT-29 cells to secrete mucus are critical mechanisms that may support the development of Akkermansia.

An effective treatment approach of liposomally encapsulated metformin in colon cancer.

Metformin is a drug that is widely used in the treatment of type-2 diabetes and its anticarcinogenic effect has been detected in many studies since the 2000s. Metformin has a short half-life and poor biocompatibility, which limits the activity of the drug. As a solution to this situation, our study aimed to increase the anticarcinogenic effects and reduce the side effects of metformin in colon cancer by liposomal encapsulation. For this purpose, in our study, liposome production was carried out using the thin film hydration method. The amount of metformin loaded in liposomes was determined by a standard absorbance curve at 237nm. Size distributions and membrane zeta potentials of the liposomes were evaluated with Malvern Zetasizer ZS90. Transmission electron microscopy was performed by staining the liposomes negatively with uranyl acetate. Cultured HT-29 cells were treated with liposomal metformin or free metformin at concentrations of 0, 10, 20, and 40mM for 24 and 48h. At the end of the treatment period, cell viability was evaluated by CellTiter-Glo luminescent cell viability test. The anticarcinogenic effects of liposomal and free metformin on HT-29 cells were compared. As a result, liposome encapsulated metformin treatment for 24h was more effective on HT-29 cells at 20- and 40-mM concentrations causing significantly greater decrease in the IC-50 dose compared to the free metformin. The result suggests that liposomal encapsulated metformin may offer a promising approach to increase the efficacy of the drug in the treatment of colon cancer.

Effects of 5-Fluorouracil on the Expression of Epigenetic Enzymes and Promoter Methylation of Selected Genes in Monolayer and Spheroid Cultures of Colorectal Cancer Cells

Background: Emerging evidence suggests that epigenetic mechanisms contribute to 5-fluorouracil (5-FU) resistance in colorectal cancer (CRC). However, there is limited research on the direct impact of 5-FU on epigenetic alterations in CRC. This study aimed to investigate how 5-FU treatment affects the expression of enzymes involved in epigenome regulation and promoter DNA methylation in human CRC cells. Methods: The viability of CRC cell lines (SW48, HCT116, LS180, and HT29) was evaluated after 48 hours of 5-FU treatment using MTT assay in both monolayer and hanging drop spheroid cultures. The cells were treated with an IC20 concentration of 5-FU and then the relative expressions of histone deacetylases (HDAC) and DNA methyltransferas1 (DNMT1) in 5-FU-treated and untreated cells were measured by quantitative RT-PCR (qRT-PCR). The status of promoter methylation of selected genes was analyzed using the methylation-specific PCR (MSP) method. Results: The 3D cultures of cells were more resistant to 5-FU than their 2D counterparts. The effect of 5-FU on HDAC1 expression was greater in 3D cultures compared to 2D cultures. 5-FU downregulated SIRT1 and DNMT1 in 2D culture of HCT116 and SW48 and upregulated them in 3D cultures of HT29 and LS180 cells. In both monolayer and spheroid cultures, 5-FU downregulated HDAC2 in HCT116, LS180, and HT29 and HDAC4 in HCT116, LS180, and SW48 cells. 5-FU primarily changed promoter methylation in monolayer cultures. Conclusion: The epigenetic response to 5-FU is cell line-specific and depends on the culture method. 5-FU modulates epigenome in CRC cells by regulating DNMT1 and HDAC expressions. 3D cultures were found to be considerably more resistant to 5-FU-induced cytotoxicity and promoter DNA methylation changes than 2D cultures. 5-FU downregulated HDAC and DNMT1, particularly in the drug-sensitive cells, and increased the levels of DNMT1 in the drug-resistant cells.

Enterococcus Induces MHC-II Expression in the Colonic Epithelium to Aggravate Graft-Versus-Host Disease

Intestinal domination by Enterococcus, a genus of Gram-positive facultative bacteria, has been associated with an increased risk of GVHD mortality in allo-HCT patients and in preclinical models. However, the immunological mechanisms underlying this association remain unclear. Several preclinical studies have demonstrated that MHC-II expression by intestinal epithelial cells (IECs) is involved in allo-antigen presentation and GVHD and can be modulated by the intestinal microbiota. MHC-II genes are constitutively expressed by professional antigen-presenting cells (APCs) but can be induced in non-professional APCs by IFN-γ. Here we reported that Enterococcus could potentially aggravate GVHD through upregulation of MHC-II expression by IECs in an IFN-γ-dependent manner. Using an MHC-disparate mouse model of GVHD (C57BL/6J into BALB/cJ), we profiled the fecal microbiome of GVHD mice by 16S rRNA sequencing and the intestinal epithelium by flow cytometry on day 7 post-transplant. We found that endogenous Enterococcus relative abundance was positively correlated with MHC-II expression by colonic IECs (CD45- CD31- EpCAM+) during GVHD ( Fig. 1a; p=0.0004; 2 replicates; n=19). To determine whether this phenotype occurs in the absence of systemic inflammation, we utilized gnotobiotic mice treated with either E. faecalis, B. producta (a bacteria genus previously associated with improved GVHD outcomes)or PBS via oral gavage. Monocolonization with E. faecalis was sufficient to upregulate MHC-II expression by colonic IECs in non-transplanted (steady-state) animals compared to PBS-treated germfree controls ( Fig. 1b; p=0.0027; 3 replicates; n=8-12/group). On the contrary, monocolonization by B. producta did not induce MHC-II expression by colonic IECs compared to germfree controls ( p=0.96). We also observed that MHC-II upregulation was promoted by specific species within the genus Enterococcus, such that compared to PBS-treated germfree controls, only E. faecalis ( p=0.0007; 3 replicates; n=8-12/group) was able to induce MHC-II expression by colonic IECs, but not E. durans ( p=0.06) , E. gallinarium ( p=0.98) and E. hirae ( p=0.99). Using a Villin-Cre IFN- γ R fl/flmouse model with genetic ablation of the IFN-γ receptor specifically in epithelial cells, we investigated whether MHC-II expression induced by Enterococcus was dependent on IFN-γ signaling. We colonized Villin-Cre pos and Villin-Cre neg littermate controls with E. faecalis via oral gavage after antibiotics decontamination (ampicillin/enrofloxacin) and profiled the intestinal epithelium. We observed that Villin-Cre posmice exhibited a significantly lower MHC-II expression in the small intestine compared to Villin-Cre negmice on day 3 post- Enterococcus colonization ( p=0.012; 2 replicates; n=7/group), suggesting that IFN-γ signaling is necessary to mediate Enterococcus interaction with the intestinal epithelium. Finally, to explore a potential intervention to reduce intestinal inflammation upon Enterococcus domination, we treated GVHD mice with either butyrate-producing B. producta or E. faecalis. Colonization with B. producta reduced fecal Enterococcus burden on day 7 post-transplant ( p=0.007; 2 replicates; n=8/group) and improved survival in GVHD mice, compared to the E. faecalis-colonized group ( p=0.002; 2 replicates; n=18-19/group). We and others have previously shown that butyrate administration can alleviate GVHD through its effects on epithelial homeostasis. To determine whether butyrate could regulate allo-antigen presentation by the intestinal epithelium, we employed the human colorectal adenocarcinoma HT-29 cell culture. We pre-treated the cells with IFNγ (100U/ml) to induce MHC-II expression, then added butyrate (5mM) to the culture for 24 hours. Butyrate treatment was sufficient to suppress MHC-II expression by HT-29 cells, compared to DMSO-treated controls ( p=0.006; 3 replicates; n=6/group). Altogether, our results suggest that one mechanism by which Enterococcus can aggravate GVHD involves the upregulation of MHC-II expression by colonic IECs, and treatment with butyrate might serve as an intervention to prevent intestinal inflammation. Further identification of the upstream mediators involved will provide additional mechanistic insight into the interaction between Enterococcus and the intestinal epithelium at steady state and during GVHD.

Diclofenac and metformin synergistic dose dependent inhibition of hamster fibrosarcoma, rescued with mebendazole

We examined whether combinig diclofenac and metformin in doses equivalent to human doses would synergize their anticancer activity on fibrosarcoma inoculated to hamsters and in vitro. Rescue experiment was performed to examine whether the prosurvival NF-κB stimulation by mebendazole can reverse anticancer effects of the treatment. BHK-21/C13 cell culture was subcutaneously inoculated to Syrian golden hamsters randomly divided into groups (6 animals per group): 1) untreated control; treated daily with 2) diclofenac; 3) metformin; 4) combinations of diclofenac and metformin at various doses; 5) combination of diclofenac, metformin and mebendazole; 6) mebendazole. Dose response curves were made for diclofenac and metformin combination. Tumor growth kinetics, biophysical, pathological, histological and immunohistochemical characteristics of excised tumors and hamster organs as well as biochemical and hematological blood tests were compared among the groups. Single treatments had no anticancer effects. Diclofenac (60 mg/kg/day) exhibited significant (P < 0.05) synergistic inhibitory effect with metformin (500 mg/kg/day) on all tumor growth parameters, without toxicity and influence on biochemical and hematological blood tests. The same results were obtained with double doses of diclofenac and metformin combination. The addition of mebendazole to the diclofenac and metformin combination rescued tumor expansion. Furthermore, diclofenac with metformin demonstrated antiproliferative effects in hamster fibrosarcoma BHK-21/C13, human lung carcinoma A549 (CCL-185), colon carcinoma HT-29 (HTB-38) and cervical carcinoma HeLa (CCL-2) cell cultures, with markedly lower cytotoxicity in the normal fetal lung MRC-5 cells. In conclusion, diclofenac and metformin combination may be recommended for potential use in oncology, due to synergistic anticancer effect in doses achievable in humans.

Different Effects of Low-Level Laser Therapy on the Proliferation of HT29 Cells in Culture and Xenograft Models.

Introduction: Different kinds of treatments have been developed to fight cancers. Low-level laser therapy (LLLT), also known as photobiomodulation therapy (PBMT), is a low-power monochromatic and coherent light that has been used successfully for healing injuries and combating malignancies. However, there are concerns about the application of LLLT to cancers due to the increased proliferation of some cancer cells after LLLT. Methods: This study investigated the effects of 650 nm and 870 nm lasers on the proliferation of HT29 colorectal cancer cell lines in vitro and in vivo. Results: The results showed that the laser with a wavelength of 870 nm did not meaningfully alter the proliferation of cultured cells. However, cell proliferation was promoted when the laser was applied within a wavelength of 650 nm. Treatment of HT29-derived tumors in nude mice with the 650 nm laser resulted in the decline of the tumor progression rate compared to controls. This result was inconsistent with the proliferative effects of the laser on the cultured cells. Conclusion: Cell behavior in response to LLLT might be different between cell culture and xenograft models.

Screening and specificity study of aptamer of common proteins in colorectal cancer

Over the years, the case fatality rate of colorectal cancer is increasing. It is increasingly important to discover new tumor markers for early diagnosis of colorectal cancer. Aptamers obtained by SELEX method can be used as molecular probes to specifically bind tumor markers. Therefore, Med-SELEX was used in this study. HT29 and SW480 cell culture medium were used as positive screen, while NCM460 cell culture medium was used as reverse screen. After 14 rounds of screening, 4 candidate aptamers were selected, which were Apt-C2, Apt-C4, Apt-C15 and Apt-C27. Their affinity to HT29 cell culture medium were 9.059 ± 2.511, 10.36 ± 3.451, 6.646 ± 1.651, 8.127 ± 1.926 nmol/L, respectively. The affinity to SW480 cell culture medium was 5.324 ± 2.341, 7.854 ± 1.611, 8.239 ± 2.666, 4.164 ± 0.9671 nmol/L, respectively. Q-PCR and quantum dot method verified the high specificity of Apt-C4. Q-PCR results showed that the average Ct value of Apt-C4 binding to colorectal cancer serum was 9.921, and the average OD value of quantum dot was 49.498. Serum stability test showed that Apt-C4 did not degrade within 60h. Therefore, Apt-C4 aptamer was confirmed to have potential as a serum probe, providing a new idea for discovering new tumor markers of colorectal cancer.

Protective effect of two Thai pigmented rice cultivars against H2O2-induced oxidative damage in HT-29 cell culture

Radicals derived from exogenous and endogenous sources are considered to be the principal cause of genetic damage. Exogenous and endogenous radicals participate in the reactive oxygen species (ROS) formation, which leads to damages in the DNA, RNA, proteins and lipids. However, dietary compounds, mainly from pigmented rice, are an essential source of antioxidants that help protect cells from damage. This study seeks to determine the antioxidant properties and cytoprotective effect of two Thai pigmented rice extracts namely the glutinous black rice (native name: Neaw dum moa37) and red rice (native name: Hom gradung-nga57) on H2O2-induced damage in HT-29 cells. The bioactive compound contents, as well as antioxidant activities of both rice extracts, were investigated. The protective effect of rice extracts on H2O2-induced damage was executed following the co-incubation method. HT-29 cells were exposed to H2O2 and different rice extract concentrations for 3 h and an MTT assay was used to measure the viability of the cell. The ROS level was determined using the 2′,7′-dichlorofluorescin diacetate (DCFDA). The result showed that glutinous black rice extract contained significantly higher contents of all analysed antioxidants and activities than red rice extract. Glutinous black rice showed a higher cytotoxic effect compared to red rice. At the non-toxic concentration of both rice extracts, the HT-29 cells were guarded against the H2O2 induced oxidative stress. Besides, the intracellular ROS accumulation result from H2O2 exposure was significantly reduced in the presence of rice extracts for both glutinous black rice and red rice compared to control. Hence, this study has demonstrated the potential properties of both pigmented rice extracts in alleviating H2O2-mediated damage in HT-29 cells.

A GdW10@PDA-CAT Sensitizer with High-Z Effect and Self-Supplied Oxygen for Hypoxic-Tumor Radiotherapy.

Anticancer treatment is largely affected by the hypoxic tumor microenvironment (TME), which causes the resistance of the tumor to radiotherapy. Combining radiosensitizer compounds and O2 self-enriched moieties is an emerging strategy in hypoxic-tumor treatments. Herein, we engineered GdW10@PDA-CAT (K3Na4H2GdW10O36·2H2O, GdW10, polydopamine, PDA, catalase, CAT) composites as a radiosensitizer for the TME-manipulated enhancement of radiotherapy. In the composites, Gd (Z = 64) and W (Z = 74), as the high Z elements, make X-ray gather in tumor cells, thereby enhancing DNA damage induced by radiation. CAT can convert H2O2 to O2 and H2O to enhance the X-ray effect under hypoxic TME. CAT and PDA modification enhances the biocompatibility of the composites. Our results showed that GdW10@PDA-CAT composites increased the efficiency of radiotherapy in HT29 cells in culture. This polyoxometalates and O2 self-supplement composites provide a promising radiosensitizer for the radiotherapy field.

Clostridium difficile toxin B induces morphological changes consistent with autophagy in the human adenocarcinoma cell line (HT-29)

Purpose: Toxin B (TcdB) is a product of the bacteria Clostridium difficile, and can trigger apoptosis or necrosis. In the present manuscript, we proposed that TcdBVPI and TcdB8864 would cause morphological changes in cultured cells that are characteristic of autophagy, a constitutive cellular event closely linked with apoptosis or necrosis.&#x0D; Methods: We examined an in vitro model of cultured HT-29 cells to determine the occurrence of autophagy. The cells were incubated separately with TcdBVPI and TcdB8864 for 2 to 24 hours, and then examined using transmission electron microscopy.&#x0D; Results: In HT-29 cells, ultrastructural changes were observed following 8 hours of treatment with TcdBVPI and TcdB8864. Upon exposure to both toxins, complex changes occurred that affected the cellular framework, including the dilated regions of the granular endoplasmic reticulum, the Golgi apparatus, as well as electron-lucent and electron-dense autophagosome-like structures. Additionally, lipid droplets were present in the cytoplasm of HT-29 cells.&#x0D; Conclusion: Our findings indicate that TcdBVPI and TcdB8864 indeed induce ultrastructural changes in HT-29 cells. The presence of these autophagic structures in the cytoplasm of the HT-29 cells suggests that autophagy may be induced during treatment by both toxins. Therefore, this effect could represent an important protective mechanism for cell survival.

Butyrate Alters Pyruvate Flux and Induces Lipid Accumulation in Cultured Colonocytes.

Butyrate is considered the primary energy source of colonocytes and has received wide attention due to its unique health benefits. Insight into the mechanistic effects of butyrate on cellular and metabolic function relies mainly on research in in-vitro-cultured cells. However, cells in culture differ from those in vivo in terms of metabolic phenotype and nutrient availability. For translation, it is therefore important to understand the impact of different nutrients on the effects of butyrate. We investigated the metabolic consequences of butyrate exposure under various culturing conditions, with a focus on the interaction between butyrate and glucose. To investigate whether the effects of butyrate were different between cells with high and low mitochondrial capacity, we cultured HT29 cells under either low- (0.5 mM) or high- (25 mM) glucose conditions. Low-glucose culturing increased the mitochondrial capacity of HT29 cells compared to high-glucose (25 mM) cultured HT29 cells. Long-term exposure to butyrate did not alter mitochondrial bioenergetics, but it decreased glycolytic function, regardless of glucose availability. In addition, both high- and low-glucose-grown HT29 cells showed increased lipid droplet accumulation following long-term butyrate exposure. Acute exposure of cultured cells (HT29 and Caco-2) to butyrate increased their oxygen consumption rate (OCR). A simultaneous decrease in extracellular acidification rate (ECAR) was observed. Furthermore, in the absence of glucose, OCR did not increase in response to butyrate. These results lead us to believe that butyrate itself was not responsible for the observed increase in OCR, but, instead, butyrate stimulated pyruvate flux into mitochondria. Indeed, blocking of the mitochondrial pyruvate carrier prevented a butyrate-induced increase in oxygen consumption. Taken together, our results indicate that butyrate itself is not oxidized in cultured cells but instead alters pyruvate flux and induces lipid accumulation.

Development of Perphenazine-Loaded Solid Lipid Nanoparticles: Statistical Optimization and Cytotoxicity Studies.

Objective Perphenazine (PPZ), as a typical antipsychotic medical substance, has the same effectiveness compared to atypical antipsychotic medications for the treatment of schizophrenia. Despite the lipophilic essence, PPZ encounters limited bioavailability caused by the first-pass metabolism following oral administration. In the present study, PPZ-containing solid lipid nanoparticles (PPZ-SLNs) were prepared and optimized based on different factors, including lipid and surfactant amount, to develop appropriate and safe novel oral dosage forms of PPZ. Methods The solvent emulsification-evaporation method was utilized to form SLNs by using soybean lecithin, glycerol monostearate (GMS), and Tween 80. Statistical optimization was done by the Box-Behnken design method to achieve formulation with optimized particle size, entrapment efficiency, and zeta potential. Also, transmission electron microscopy, in vitro release behavior, differential scanning calorimetry (DSC), and powder X-ray diffractometry (P-XRD) studies and cytotoxicity studies were assessed. Results Optimization exhibited the significant effect of various excipients on SLN characteristics. Our finding indicated that the mean particle size, zeta potential, and entrapment efficiency of optimized PPZ-SLN were, respectively, 104 ± 3.92 nm, −28 ± 2.28 mV, and 83% ± 1.29. Drug release of PPZ-SLN was observed to be greater than 90% for 48 h that emphasized a sustained-release pattern. The DSC and P-XRD studies revealed the amorphous state of PPZ-SLN. FTIR spectra showed no incompatibility between the drug and the lipid. Performing cytotoxicity studies indicated no significant cytotoxicity on HT-29 cell culture. Conclusion Our study suggests that PPZ-SLNs can make a promising vehicle for a suitable therapy of schizophrenia for the oral drug delivery system.

Heat-Treated Bifidobacterium longum CECT-7347: A Whole-Cell Postbiotic with Antioxidant, Anti-Inflammatory, and Gut-Barrier Protection Properties.

Non-viable preparations of probiotics, as whole-cell postbiotics, attract increasing interest because of their intrinsic technological stability, and their functional properties, such as immune system modulation, gut barrier maintenance, and protection against pathogens. However, reports on Bifidobacteria-derived postbiotics remain scarce. This study aims to demonstrate the functional properties of a heat-treated (HT), non-viable, Bifidobacterium longum strain, CECT-7347, a strain previously selected for its anti-inflammatory phenotype and ability to improve biomarkers of intestinal integrity in clinical trials. The study used the nematode Caenorhabditis elegans and HT-29 cell cultures as eukaryotic model systems. Our results show that HT-CECT-7347 preserves the capacity to protect against oxidative stress damage, while it also reduces acute inflammatory response and gut-barrier disruption, and inhibits bacterial colonization, by activating pathways related to innate immune function. These findings highlight the interest of the ingredient as a novel postbiotic and pave the way to broaden the range of HT-CECT-7347 applications in gut health.

Supercritical CO2 Extraction and Microencapsulation of Lycopene-Enriched Oleoresins from Tomato Peels: Evidence on Antiproliferative and Cytocompatibility Activities.

Tomato peels are used as a valuable material to extract lycopene-rich oleoresins by supercritical CO2 extraction. The extraction involves continuous circling of CO2 to the extractor after removing the solute in the separators, S40 and S45, where the solvent power of the CO2 is reduced by reducing pressure down to 20 MPa in S40 and 5 MPa in S45, respectively, leading to two extracts. Lycopene is found to be the major compound, representing 93% and 76% of the total carotenoids in S40 and S45 extracts, respectively. The two extracts are microencapsulated in whey protein concentrate and acacia gum by complex coacervation and freeze-drying, leading to corresponding P40 and P45 powders, with antioxidant activity of 8.57 ± 0.74 and 9.37 ± 0.48 mMol TEAC/g DW in P40 and P45, respectively. Different structural and morphological patterns are observed, with finer microparticles of 1–2 µm in P45. Both powders show dose and time-dependent antiproliferative activity. The half-maximal inhibitory concentration values are 100 µg/mL for P40 and 750 µg/mL for P45 sample, indicating a higher antiproliferative effect of P40 over P45 in HT-29 cell culture. The powders have an extended range of cytocompatibility, up to 1000 µg/mL, in L929 normal cells, stimulating the cell growth. Lycopene retention is tested, and values of 48% and 29% in P40 and P45 are found after 21 days at 25 °C, with the degradation rate in P45 significantly higher, due to the higher content of the surface lycopene, which favored its degradation.

Development of a CD63 Aptamer for Efficient Cancer Immunochemistry and Immunoaffinity-Based Exosome Isolation.

CD63, a member of transmembrane-4-superfamily of tetraspanin proteins and a highly N-glycosylated type III lysosomal membrane protein, is known to regulate malignancy of various types of cancers such as melanoma and breast cancer and serves as a potential marker for cancer detection. Recently, its important role as a classic exosome marker was also emphasized. In this work, via using a magnetic bead-based competitive SELEX (systematic evolution of ligands by exponential enrichment) procedure and introducing a 0.5 M NaCl as elution buffer, we identified two DNA aptamers (CD63-1 and CD63-2) with high affinity and specificity to CD63 protein (Kd = 38.71 nM and 78.43, respectively). Furthermore, CD63-1 was found to be efficient in binding CD63 positive cells, including breast cancer MDA-MB-231 cells and CD63-overexpressed HEK293T cells, with a medium binding affinity (Kd ~ 100 nM) as assessed by flow cytometry. When immunostaining assay was performed using clinical breast cancer biopsy, the CD63-1 aptamer demonstrated a comparable diagnostic efficacy for CD63 positive breast cancer with commercial antibodies. After developing a magnetic bead-based exosome immunoaffinity separation system using CD63-1 aptamer, it was found that this bead-based system could effectively isolate exosomes from both MDA-MB-231 and HT29 cell culture medium. Importantly, the introduction of the NaCl elution in this work enabled the isolation of native exosomes via a simple 0.5M NaCl incubation step. Based on these results, we firmly believe that the developed aptamers could be useful towards efficient isolation of native state exosomes from clinical samples and various theranostic applications for CD63-positive cancers.

Anti-Mutagenicity and Apoptotic Effects of Teucrium polium L. Essential Oil in HT29 Cell Line

Objectives: Regarding the high prevalence of cancer in Iran and the cytotoxic properties of T. polium, the current study aimed to investigate the cytotoxic and anti-mutagenicity effect of T. polium essential oil (TpEO) on human colon adenocarcinoma cell line (HT29). Methods: HT29 cells were cultured in L-glutamine, RPMI Sigma (1640), with 10% of FBS (fetal bovine serum). Then, the cultures were incubated with 5% CO2 at 37°C, and MTT assay was used to recognize cell proliferation under the inhibitory influence of TpEO. The cell cycle progression was monitored by Sub-G1 apoptosis assay using flow cytometry. Eventually, the anti-mutagenicity property was evaluated using the Ames test employing TA100 and exposure to sodium azide as the carcinogenic substance. Results: The cytotoxic effect of TpEO on HT29 cells was 66.867 ± 1.37 µg/mL. Cultured HT29 cells treated with TpEO exhibited morphological features of apoptosis. TpEO preventive effect was about 78.18%. Conclusions: This study showed that TpEO may be useful for treating colon cancer.

High-throughput screening reveals higher synergistic effect of MEK inhibitor combinations in colon cancer spheroids

Drug combinations have been proposed to combat drug resistance, but putative treatments are challenged by low bench-to-bed translational efficiency. To explore the effect of cell culture format and readout methods on identification of synergistic drug combinations in vitro, we studied response to 21 clinically relevant drug combinations in standard planar (2D) layouts and physiologically more relevant spheroid (3D) cultures of HCT-116, HT-29 and SW-620 cells. By assessing changes in viability, confluency and spheroid size, we were able to identify readout- and culture format-independent synergies, as well as synergies specific to either culture format or readout method. In particular, we found that spheroids, compared to 2D cultures, were generally both more sensitive and showed greater synergistic response to combinations involving a MEK inhibitor. These results further shed light on the importance of including more complex culture models in order to increase the efficiency of drug discovery pipelines.

The genes CNV and expression as a factors of HT-29 cells radioresistance.

e15590 Background: The initial radioresistance of tumor cells associated with certain molecular genetic features has a great influence on the effectiveness of radiation therapy (RD). These features include the transcriptional activity of genes that regulate DNA repair, cell cycle and apoptosis, and their copy number variation (CNV). The aim of the study was to analyze the expression and CNV of genes that regulate DNA repair, cell cycle and apoptosis in HT-29 cells subjected to RD. Methods: HT-29 cell culture was used in the study. For the model experiment, doses of 5 and 7 Gy were used (irradiation was performed 5 times every 24 hours on a Novalis TX linear accelerator). Cell counting was carried out in Goryaev chamber (0.4% trypan blue solution). On the 5th day of irradiation, HT-29 was removed from the substrate with Trypsin / Versen solution. RNA was isolated according to the method of Chomczynski&amp;Sacchi. For cDNA synthesis was used a "REVERTA-L". reagent. The phenol-chloroform method was used to DNA isolate. The RT-qPCR method was used to determine the CNV and expression of 32 genes: AKT, ATM, BRIP, BRCA1, BRCA2, CDK1, CDKN1B, CCND1, CCND3, EXO1, FGFR2, HIST1, H2AX, KU70, PTEN, RAD50, RAP80, RIF1, RNF8 TOPB1, TP53, XRCC4, BAX, CASP8, CASP3, CASP9, MDM2, BCL2, RBBP8, EP300, LIG4, C-FLIP. Statistical analysis was performed using ANOVA and Spearman's rank correlation coefficient (r). Results: After irradiation, only a specific pool of HT-29 cells remained viable: 32% for 5 Gy and 20% for 7 Gy of the initial number of cells. In HT-29 cells irradiated with 7 Gy, the CNV of the BRCA2, H2AX, CASP9 and RBBP8 genes was increased (p &lt; 0.05) and the CNV of BCL2 gene was reduced (p &lt; 0.05) relative to intact cells. In cells irradiated with 5 Gy only CASP9 and RBBP8 CNV was statistically significantly (p &lt; 0.05) increased. In cells irradiated with 5 and 7 Gy, expression of BRCA2, H2AX, CASP9 and RBBP8 genes was statistically significantly (p &lt; 0.05) increased and the expression of the BCL2 gene was reduced relative to intact cells. A strong positive correlation was observed between CNV and expression of the studied genes: r = 0.998 for control, r = 0.989 for cells irradiated at 5 Gy, and r = 0.993 for cells irradiated at 7 Gy. Conclusions: The study showed that 5 day RD at 5 and 7 Gy leads to selective survival of 32% and 20% of cells, respectively, with increased CNV and expression of BRCA2, H2AX, RBBP8 CASP9 genes and reduced CNV and expression of BCL2 gene.

In vitro/in vivo assessment of the targeting ability of [99mTc] Tc-labeled an aptide specific to the extra domain B of fibronectin (APTEDB) for colorectal cancer.

The APTEDB is an aptide specific to the extra domain B (EDB) of fibronectin with high affinity for EDB, which is expressed in malignant tumors including brain cancer (U87MG) and colorectal cancer (HT-29). Aim of this study was to evaluate the [99mTc] Tc-APTEDB potential as an imaging probe for colorectal cancer. Radiochemical purity was evaluated by HPLC and radio-isotope TLC scanner. Blocking study for specific binding assay and affinity calculation (Kd) on HT-29 cell lines were also carried out. Planar imaging and bio-distribution studies were performed in HT-29 tumor-bearing mice. The APTEDB was efficiently labeled with technetium-99m in high radiochemical yield (up to 97%). Cellular binding study demonstrated specific binding of the [99mTc] Tc-APTEDB in cultured HT-29 cells. The Kd value was found to be 40.46 ± 13.39nM. The tumor-to-muscle ratio was ~ 1.5 in ex vivo bio-distribution study at 1h after injection. Planar imaging study showed higher activity accumulation in EDB expressing HT-29 tumor relative to muscle (used as control) (~ 1.7) at 1h. Although more studies are required to find out the full potential of this radio-ligand as an imaging probe, the present results nevertheless provide useful information about [99mTc] Tc-APTEDB, which might be beneficial in design and development of new [99mTc] Tc-APTEDB for efficient targeting of tumor in vivo.

Evaluation of cell-penetrating peptide-peptide nucleic acid effect in the inhibition of cagA in Helicobacter pylori.

Helicobacter pylori is the most common cause of chronic infection in human and is associated with gastritis, peptic ulcer disease, and adenocarcinoma of mucosa-associated lymphoid tissue cells. Peptide nucleic acid (PNA) is a synthetic compound, which can inhibit the production of a particular gene. This study aimed to investigate the effect of PNA on inhibiting the expression of cagA. After confirmation of the desired gene by polymerase chain reaction (PCR), the antisense sequence was designed against cagA gene. The minimum inhibitory concentrations of conjugated PNA against H. pylori was determined. The effect of the compound on the expression level of the cagA was investigated in HT29 cell culture using real-time PCR. The results showed 2 and 3 log reduction in bacterial count after 8- and 24-h treatment with 4 and 8 μM of the compound, respectively. The lowest expression level of the cagA gene was observed at a concentration of 8 μM after 6 h. The results of this study showed that cell-penetrating peptide antisense can be employed as effective tools for inhibiting the target gene mRNA for various purposes. Moreover, further research is necessary to assess the potency, safety, and pharmacokinetics of CPP-PNAs for clinical prevention and treatment of infections due to H. pylori.